Recent progress in this laboratory now makes it possible to attack some of the central questions of protein stability and function. How do weak bonding interactions form and stabilize protein structures? How do native proteins regulate their functions by making and breaking subsets of these same interactions. At present we know little about the parameters and principled involved. Advances in hydrogen exchange (HX)-based methods are uniquely able to probe these issues by resolving and measuring structural free energies and free energy changes. In the previous project period, we demonstrated the design and synthesis of a synthetic, stable, alpha-helical peptide that can see as a carrier onto which bonding interactions of various kinds can be built. We showed that the stabilization free energy of experimentally imposed interactions and their sites of action can then be determined from the NMR-detected hydrogen exchange behavior of the various peptide group NHs. We propose to use this system to evaluate, in a controlled context, the free energy of the many kinds of bonding interactions that proteins use to form and stabilize their structures. Proteins regulate their functions through structure change, i.e. by manipulating specific bonding interactions. For example, the archetypal allosteric protein, hemoglobin, controls the oxygen affinity of its heme groups by using part of the binding energy of its initial oxygen ligands to break bonding interactions that selectively stabilize its law affinity, T state. To understand how this energy interconversation process works, it will be necessary to locate the detailed changes in structure, measure their individual free energies, and quantify in free energy terms - how the different changes interact, how they add and subtract and transfer, to constitute the allosteric function. In the previous project period we demonstrated the unique ability of hydrogen exchange methods to locate and quantify site-resolved free energy and free energy, changes in hemoglobin. This work was limited by our available methodology to a small but important fraction of the protein. Work was done to develop the next generation of hydrogen exchange methods, involving both mass spectrometry and NMR, that will extend our HX labeling technology to the whole protein. We propose to complete this development and then to exploit these methods to learn how the allosteric machinery functions.